In this R21 application we postulate that the broadly HIV neutralizing monoclonal antibodies 2F5 and 4E10, against the membrane proximal external region (MPR) of GP41 were induced in the donors by MPR epitopes that had been post-translational modified. Our high-risk idea is to generate MPR immunogens incorporating chemically-modified amino acid residues that reflect the post-translational modifications that can occur during viral infections. These modifications are immediately adjacent to the MPR epitopes recognized by 2F5 and 4E10. We believe these novel immunogens will enhance the potency of existing epitopes and/or 'unmask' epitopes previously hidden from the immune system that induce neutralizing antisera. The idea to use such peptides containing the modified residues is unique. MPR immunogen design has focused on eliciting antibody responses that cross-react with phospholipids because the broadly neutralizing mAbs 2F5 and 4E10 exhibit unusual cross-reactivity with lipids. This led many investigators to attribute their dual affinity for both gp41 and phospholipids in the viral membrane as the source of their potency. However, the lipid reactivity of these antibodies is primarily directed at negatively charged lipid head groups, which are chemically similar to phosphorylated or nitrated amino acids. Thus, amino acid modifications, not lipids, may be the true cause of the unusual dual recognition properties of 2F5 and 4E10. We will synthesize MPR peptide sequences containing nitrated, phosphorylated, or non-hydrolyzable charge equivalent, phosphonated or sulfonated residues, attach them to lipid anchors, characterize their secondary structure and measure their partition coefficient in lipid vesicles. The lipopeptides will be formulated in an immunogenic liposome and used to immunize mice and rabbits. Antisera will be tested for antibody titers using a validated ELISA and neutralization will be measured in a clinically relevant PBMC HIV infection assay. To learn if presentation of the modified epitopes in a viral-mimic triple helix bundle increases immunogenicity and/or results in the generation of neutralizing antibodies, the modified peptides will be incorporated into a scaffold that presents three epitopes on the same lipid anchor and evaluated as described for the lipopeptides. To determine if the immunogenicity of the modified epitopes requires a lipid anchor, we will replace the lipid on the modified epitopes with polyethylene glycol and repeat the in vitro characterization and in vivo immunization studies. Results from these experiments will guide subsequent rounds of epitope design to optimize antisera titers and HIV neutralization activity. Success in generating neutralizing antibodies from chemically-modified peptide epitopes would enable structural studies on the epitope-antibody complex and lead to protection studies in primate HIV challenge models. This unusual approach could provide a widely applicable epitope design strategy for development of vaccines to prevent infectious disease such as HIV.